S. mutans and A. naeslundii. Experimental materials containing Cu-MBGN showed a dose-dependent Cu release with an initial burst and a further increase after 28 days. The composite containing 10% Cu-MBGN had the best anti-bacterial effect on S. mutans, as evidenced by the lowest adherence of free-floating bacteria and biofilm formation. In contrast, the 45S5-containing materials had the highest S. mutans adherence. Ca release was highest in the bioactive control containing 15% 45S5, which correlated with the highest number of open porosities on the surface. Polymerization shrinkage was similar for all tested materials, ranging from 3.8 to 4.2%, while the degree of conversion was lower for Cu-MBGN materials. Cu-MBGN composites showed better anti-bacterial properties than composites with 45S5 BG.Experimental dental resin composites containing copper-doped mesoporous bioactive glass nanospheres (Cu-MBGN) were developed to impart anti-bacterial properties. Increasing amounts of Cu-MBGN  were added to the BisGMA/TEGDMA resin matrix containing micro- and nano-fillers of inert glass, keeping the resin/filler ratio constant. Surface micromorphology and elemental analysis were performed to evaluate the homogeneous distribution of filler particles. The study investigated the effects of Cu-MBGN on the degree of conversion, polymerization shrinkage, porosity, ion release and anti-bacterial activity on  Dental caries is one of the most common dental diseases affecting more than one-third of the population worldwide . RecentlResearchers are incorporating compounds such as amorphous calcium phosphate (ACP) and bioactive glass (BG) to counter these challenges in dental resins. BG has attracted much attention in the field due to its flexible composition, which can be adjusted to attain a desired biological and remineralization response. Some studies have shown that incorporating ACP ,7,8,9 anCommercially available composites use silane coupling agents to improve mechanical strength and prevent crack propagation after the material has been subjected to high stress. Silane coupling molecules are bifunctional. They form a covalent bond (Si-O-Si) with the filler particles on one side and simultaneously copolymerize with the methacrylate group in the resin phase. This helps to prevent the propagation of cracks and increases the mechanical strength of resin composites . HoweverCopper is well known for its multifunctional biological activities . DependiBinary filler blend consisting of Ba-glass microfillers as a base and 10 wt% of either Cu-MBGN, silica nanofillers, or 45S5 BG with a total filler amount of 65 wt%.Ternary filler blend consisting of Ba-glass microfillers as a base and a combination of 1, 5 or 10 wt% Cu-MBGN with silica filler up to 15 wt%, making a total filler amount of 70 wt%. As a bioactive control, 15 wt% 45S5 was added to the Ba-glass microfillers (15-BG) and as an inert control, 15 wt% silica fillers were added to the filler base of Ba-glass microfillers.While previous studies focused on mechanical  and polyTherefore, we hypothesized that incorporating Cu-MBGN or the combination of Cu-MBGN and silica nanofillers into the resin matrix could reduce polymerization shrinkage, achieve anti-bacterial properties, reduce biofilm formation, and promote remineralization. In order to verify the hypothesis, we evaluated the micromorphology and uniform dispersion of filler particles in the resulting experimental composites by scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDX). The effect of resin composites with Cu-MBGN on polymerization  was investigated using attenuated total reflectance accessory on a Fourier transform infrared spectroscopy (ATR-FTIR) and micro computed tomography (micro-CT). Remineralizing and anti-bacterial properties were estimated from the release of calcium and copper ions by inductively coupled plasma mass spectrometry (ICP-MS), while biofilm formation and anti-bacterial response to free-floating cariogenic bacteria were investigated using anti-bacterial assays.Cu-MBGN glass particles were prepared as mentioned previously in Zheng et al. . The den2) was used to cure the experimental resin composite three times on each side for a total of six times and 120 s. Before removing from the mold, experimental resin composites were polished on both sides. They were then stored at room temperature in a sterile well plate.Custom-made Teflon mold with 2 mm diameter and 3 mm height was filled with experimental resin composite. The experimental resin composites were placed in the mold using a stainless steel Heidemann spatula and round condenser, and trapped air was removed while applying gentle pressure. Excess material was also removed. Bluephase PowerCure  was used to calculate the shrinkage, volume of the entire cylinder of resin and to visualize the volumetric difference between pre- and post-curing of the composites.To calculate the shrinkage caused by polymerization, samples were scanned both before and after light curing. Briefly, for each sample, a stopper was placed in the Kapton tube on top of the micro-CT specimen holder, and the experimental resin composite was transferred into a Kapton tube (2 mm diameter and 3 mm height) using a stainless steel Heidemann spatula and round condenser. Entrapped air was removed by pressing with light pressure, and the extra material was removed from the tube. The holder with the experiment sample was then transferred to the micro-CT and scanned using a desktop micro-CT scanner . The samples were scanned at 50 kV, 190 uA, at a pixel size of 3.9 µm and a rotation step of 0.79−1 and aromatic C···C band at 1608 cm−1 acquired from FTIR spectrum [Attenuated total reflection Fourier transform infrared spectrometry  was used to analyze the percentage of reacted methacrylate groups (degree of conversion) of each material on the sample surface. Three measurements per group were performed (n = 3). The degree of conversion was calculated according to the “two-frequency baseline” method, where the percentage of unreacted methacrylate groups in the material is determined by using the ratio of peak intensities of aliphatic C=C mode at 1638 cmspectrum .DC (%)=Nano-CT was performed to study the porosity of the samples at a sub-micrometer resolution. Following the shrinkage study, one cured sample from each group was randomly selected and scanned using a nano-CT system . The images were acquired at a final isotropic resolution of 1.3 µm per pixel, at camera binning = 1 × 1, at 70 kV accelerating voltage, 210 μA current, and with a 0.125 mm aluminum filter placed in front of the camera. The samples were rotated 360° about their vertical axis using a step size of 0.37°, and the exposure time was set to 1100 ms per projection. An average of 4 frames was taken, the total being 4400 ms per projection. Images were reconstructed using NRecon software  with a filtered back-projection algorithm with ring artefact correction of 9 and beam hardening correction of 60%. CTan  was used to carry out the data analysis of the reconstructed 3D images. The pores were segmented, and porosity and pore morphometry were calculated from the segmented pores. For visualization, Dragonfly software ver 3.6 was used.v/v) single distilled nitric acid (HNO3) was added to each tube and kept at 4 °C. ICP-MS determined ionic concentrations of supernatant.Following micro-CT, we measured the ion release behavior of our scanned samples using an inductively coupled plasma mass spectrometer  equipped with a Cetac ASX-250 autosampler and an ESI OneFAST sample introduction system. Briefly, an immersion solution of 50 mmol/L 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)  and 133 mmol/L NaCl  was prepared in deionized water. Each sample was incubated at 37 °C on a plate shaker in the dark with 2 mL of immersion solution in a 15 mL propylene tube. For a total of 28 days, the samples were kept immersed. Fresh immersion solution was added when solutions from samples were collected on day 1, day 3, day 7, day 14 and day 28. In order to keep the supernatant for ICP-MS mass spectrometry analysis, 1%  was run as an unknown. Concentrations of boron, calcium and copper were determined by analysis of masses 11, 44 and 65, respectively. Scandium (mass 45) was used as an internal standard for all measured elements such as boron (B), calcium (Ca), and copper (Cu), After the ion release, samples were analyzed to study the composite matrix’s surface morphology and ion distribution. Samples were fractured, and one section from each sample was randomly selected to study the morphology by scanning electron microscopy (SEM) . Elemental analysis for elemental distribution on the composites was performed by energy-dispersive X-ray spectroscopy (EDX) with the aforementioned apparatus. Elemental composition was recorded, and average values were calculated. The same setup was repeated for all the samples from the ion release test.Streptococcus mutans ATCC 35668 or Actinomyces naeslundii ATCC 19039, Manassas, VA, USA). Samples were placed in sterile Eppendorf tubes after being sterilized by UV  at Energy 25 (2500 µJ/cm2) for 5 min on each side (10 min UV). S. mutans was cultured on brain heart infusion (BHI) agar at 37 °C and 5% CO2 for 24 h. Bacteria were inoculated in BHI medium and grown at 37 °C and 5% CO2 for 24 h. The overnight culture was diluted 100-fold in fresh BHI and grown at 37 °C and 5% CO2 to an optical density (OD600) of 0.6–0.7. A. naeslundi was cultured on BHI agar in an anaerobic atmosphere  for 72 h before inoculation in 10 mL fresh BHI and growth to obtain OD600 = 0.6–0.7.We studied the biofilm formation and anti-bacterial activity on the surface of the experimental resin composites using bacterial strains  and bacteria attached to the experimental resin composite (biofilm) were evaluated by counting the number of colony-forming units (CFU) (n = 3). Briefly, an aliquot of the bacterial suspension was removed, and the samples were washed quickly 3 times in sterilized distilled water and transferred to 1 mL fresh BHI. Bacteria attached to the samples were collected by dispersing the biofilm by vortexing for 1 min at 2400 rpm. Detached bacteria from biofilm and free-floating bacteria were serially diluted, spotted on the blood agar plates and incubated for 48 h at 37 °C and 5% CO2. Finally, colonies were manually counted.100 µL of bacterial suspension was added to each sample and incubated for 24 h at 37 °C and 5% COt-test. GraphPad Prism 8  was used to conduct all analyses. The significance level was set to 0.05, 0.01 or 0.001.A Kolmogorov–Smirnov test was used to determine if the dataset’s distributions were parametric or nonparametric. After that, a normality test was performed (Holm-Sidak method). The data were given as arithmetic mean values with standard deviation when normally distributed. Regular ANOVA was used for the rest of the analysis, with a Tukey test for post-hoc comparison. All the bacterial datasets were tested with a two-tailed student Surface morphologies of binary experimental composites , ternaryThe atomic percentage of Si was above 92% in experimental composites, which was significantly higher in ternary composites than the 15-BG composite and was at the same level as Si composites A. The amOn the other hand, the amount of Cu identified on the surface of experimental composites was directly proportional to the percentage of Cu-MBGN incorporation C. It wasWe studied the volumetric polymerization shrinkage in experimental resin composites using micro-CT. The results  showed tIn order to assess more detailed descriptions on where in the composite the shrinkage took place, composites where scanned pre- and post-curing without removal from the nano-CT . The genTotal porosity results A indicatWe observed a high percentage of open porosity B in 15-BIn contrast, the percentage of closed porosity D appeareThe degree of conversion of all the composite samples was over 70%, except 10-CuBG (63%) and 10-CuBG-Si (56%) . Both 10Ion concentration curves demonstrating the ion release profile at each time point and cumulative mass release of B, Ca and Cu during the observation period of 28 days are shown in The release of B from the 10-Si and 15-Si composites continuously increased during the 28 days. However, it was higher in 15-Si. The concentrations of Ca and Cu B,E from The release of B and Ca ion concentration was lower in experimental composites compared to 15-BG, but no dose-dependent effect on release was observed. However, the trend of B ion release was similar in the experimental groups: initial high release followed by a minor decrease and then a gradual increase over a defined period. The concentration of B remained the lowest in 10-CuBG compared to other experimental composites. Experimental composites with a low Cu-MBGN concentration (1-CuBG-Si and 5-CuBG-Si) showed the same pattern of Ca release; Ca ions were released in a burst on day 1 (higher in 5-CuBG-Si), but Ca ions release drastically dropped on day 3, followed by a gradual decrease. Whereas in experimental composites with a high concentration of Cu-MBGN (10-CuBG and 10-CuBG-Si), Ca ions were released in a burst on day 1, but their release decreased drastically on day 3, followed by a gradual increase on the respective time points.In contrast, the Cu ions concentrations for experimental composites were superior to those of the BG and Si-composites E. A doseStreptococcus mutans (S. mutans) and Actinomyces naeslundii (A. naeslundii)  than in 15-Si.We investigated the anti-bacterial effect of the composites and biofilm formation by slundii) . The antmposites A. 10-CuBS. mutans attachment, respectively, compared to 5-CuBG-Si. The attachment of S. mutans was significantly lower in 10-CuBG-Si compared to 1-CuBG-Si (p < 0.05).On the other hand, biofilm formation in both the binary and ternary groups was observed to be higher in BG composites than in other groups B. The amA. naeslundii  and the same 10% filler amount of either inert , bioactive control  or experimental fillers .SEM-EDX showed a homogeneous distribution of the fillers for all materials of the binary filler group. Well-blended and uniformly distributed fillers are a prerequisite for any experimental composite, especially if nanoscale fillers are included in the composition. Nanofillers increase the viscosity of the composite and severely limit the maximum filler amount due to the large surface area. The mesoporous particles used in this study have a much larger diameter than the silica nanofillers . However, the surface area of Cu-MBGN reaches almost twice that of silica nanofillers due to surface porosity , making 50 diameter is 4 µm but can reach 13 µm (d99). Phosphorus also predominates in 10-BG compared to the other two materials in the binary filler group, as 6% P2O5 is present in the structure of 45S5 BG. Despite the absence of P in 10-CuBG and 10-Si, traces of P were most likely observed due to immersion in the HEPES-buffered solution. Similarly, 10-CuBG has the highest amount of Cu, which should not be present in 10-BG and 10-Si. However, it is detected by EDX and in the ion release study with a highly sensitive ICP-MS instrument. A possible origin of Cu in non-Cu MBGN materials is the stainless steel instruments used to make the composite samples.Agglomerates visible in 10-BG are the only non-uniform surface morphology. Agglomerates of 5–10 µm B with hiWhen immersed in simulated body fluid (SBF), free Cu-MBGN particles release most Cu and Ca ions in the first 24 h . This beS. mutans and A. naeslundii. Similar to 10-CuBG, a reduction in free-floating bacteria was also observed with 10-BG, but a 0.99-fold higher amount of biofilm formed on the surface of 10-BG than on the inert control 10-Si. The anti-bacterial effect of 10-BG derives from a conventional 45S5 BG, which causes alkalinity of the immersion medium resulting from the release of OH ions from 45S5 BG [S. mutans is considered a successful colonizer and a highly competitive and prosperous bacterium in dental biofilms on caries-affected dental hard tissue surfaces. Its acidogenicity and acidurance are most likely responsible for S. mutans being one of the main bacterial species responsible for the occurrence of dental caries [A. naeslundii, on the other hand, is a pioneer bacteria in oral biofilm formation and has been frequently found in cervical caries, but is less dominant in the microbiome of mature dental plaque [A. naeslundii than a strong antimicrobial action on S. mutans, the preventive effect of 10-CuBG on secondary caries is still adequate.This study shows the secondary jump in Ca and Cu ion release from 10-CuBG at 28 days. In a clinical setting, this property could be advantageous as the initial Cu release could immediately act against bacteria remaining in the tooth cavity after preparation and later intercept bacterial adhesion and biofilm formation after restoration placement, thus preventing secondary caries . Confirm 45S5 BG ,50,51,52 45S5 BG . Informal caries ,55. A. nl plaque . A multil plaque ,58,59. SVolumetric polymerization shrinkage was in a narrow range of 3.8% to 4.2% for all the composites tested in this study, slightly higher than for sculptable commercial composites (2% to 3%) and lower than values reported for flowable composites (5%) . AlthougThe ternary filler group investigated the dose-dependent effect of the combined action of 1, 5 or 10 wt% Cu-MBGN and silanized silica up to 15%  with the Ba glass microfiller base. This group was also included in the bioactive control with 15 wt% of 45S5 BG (15-BG) and the inert control with 15 wt% silica nanofillers.Like in the binary filler group, SEM inspection of the micromorphology of the ternary group also showed a uniform distribution of fillers, revealing a denser structure on the 10-CuBG-Si than on 1-CuBG-Si or 15-Si. Due to the largest share of Cu-MBGN particles and silica nanoparticles, this was also macroscopically the most viscous material that required strong condensing. Consequently, this composite was one of the least porous materials in this study. The percentage of total porosity decreased with the increasing amount of Cu-MBGN and thus increased the CuBG composites’ viscosity. The porosity is one of the rarely investigated material properties strongly influencing composite materials’ physical and mechanical properties, affecting the crack initiation and directing the crack propagation upon exposure to high load . The lacRegarding ion release, increased porosity enhances ion release from bioactive components in composite materials . In contS. mutans is also confirmed for the experimental CuBG composites. The dose-dependent Cu concentrations in the immersion solutions were inversely related to the number of free-floating S. mutans. The formation of monospecies biofilms on the surface of the CuBG composites was also reduced. On the other hand, a higher number of S. mutans CFU was found on the surface of 15-BG. The increased percentage of open pores in 15-BG probably increased the surface roughness of the material, which might have facilitated bacterial attachment [The anti-bacterial effect of Cu on tachment ,64. Antitachment . They arThe current composition of CuBG composites apparently did not result in the desired reduction in polymerization shrinkage, despite the reduction in the degree of conversion. However, the degree of conversion decreased with the increase of the Cu-MBGN content in the ternary filler group. This decrease is probably related to the high viscosity already mentioned. The increased surface area of the mesoporous particles and the presumed presence of supraparticles in the 10-CuBG-Si could be responsible for light attenuation and lower light transmittance . ConsequS. mutans and significantly reduced the formation of a S. mutans biofilm. Further fine-tuning of the resin matrix and resin/filler ratio is needed to achieve lower polymerization shrinkage and higher conversion efficiency. Considering the results of our previous studies, we could conclude that the material with the combination of 5 wt% copper-doped mesoporous bioactive glass and silica nanofillers has appropriate mechanical properties, polymerization efficiency, and ion-releasing and anti-bacterial properties.This study showed that the anti-bacterial effect of copper-doped mesoporous bioactive glass nanospheres was maintained when they were embedded in a dental resin matrix with other inert fillers. The release of calcium and copper ions was sustained and detectable until the end of the measurement (28 days). The experimental composites had an anti-bacterial effect against free-floating


Simplifying device layout, particularly avoiding the complex fabrication steps and multiple high-temperature treatment requirements for electron-selective layers (ESLs) have made ESL-free perovskite solar cells (PSCs) attractive. However, the poor perovskite/substrate interface and inadequate quality of solution-processed perovskite thin films induce inefficient interfacial-charge extraction, limiting the power conversion efficiency (PCEs) of ESL-free PSCs. A highly compact and homogenous perovskite thin film with large grains was formed here by inserting an interfacial monolayer of diethanolamine (DEA) molecules between the perovskite and ITO substrate. In addition, the DEA created a favorable dipole layer at the interface of perovskite and ITO substrate by molecular adsorption, which suppressed charge recombination. Comparatively, PSCs based on DEA-treated ITO substrates delivered PCEs of up to 20.77%, one of the highest among ESL-free PSCs. Additionally, this technique successfully elongates the lifespan of ESL-free PSCs as 80% of the initial PCE was maintained after 550 h under AM 1.5 G irradiation at ambient temperature. PSCs arectrodes ,15,16,17ectrodes ,19. In tectrodes ,21.2 ESL. Since it is known that TiO2 causes the perovskite layer to decompose by creating oxygen vacancies, the removal of the TiO2 ESL is mostly responsible for the high device stability [The subsequent focus in PSCs is on the simplified fabrication technique that offers simple and inexpensive mass production in order to become economically relevant in the context of high efficiency and reasonable stabilities . In thistability . Howevertability ,26,27. Atability ,29.3 irradiation) to prevent carrier recombination in the perovskite or at the TCO/perovskite interface [The efficacy of the resulting ESL-free PSCs is enhanced by carefully adjusting the compositions, architecture, and morphology of the perovskite layer as well as pre-treating the TCO -surface modification with DEA to enhance the ITO/perovskite interaction and perovskite crystallization. Because amine groups typically interact with the ITO surface and Pb-based substances are reported to link with –OH groups, we chose DEA, which contains both –OH and amine groups, as the contact modifier between ITO and perovskite . As anti2. This solution was stirred for 4 h in a glove box. Spiro-OMeTAD was dissolved in chlorobenzene at a concentration of 80 mg/mL to make the precursor for the hole-selective layer. Tert-butylpyridine (28.5 μL in 1 mL of chlorobenzene) and lithium bis-(trifluoromethanesulfonyl) imide (8.75 mg/mL) were then added as additives, and the mixture was stirred in a glove box for 6 h.Perovskite solution was obtained by mixing anhydrous dimethylformamide/dimethylsulfoxide (600 mg/78 mg) with 0.15 M of formamidinium lead iodide (FAI), 0.85 M of methylammonium lead iodide (MAI), and 1.025 M of PbIThe ITO substrates were ultrasonically cleaned with detergent solution, deionized water, acetone, ethyl alcohol, and deionized water for 20 min, respectively. The ITO substrates were exposed to UV-ozone for 20 min after drying. The cleaned ITO substrates were spin-coated with the DEA pure solvent for 30 s at 2000 rpm and then dried for 10 min at 100 °C. The bare cleaned ITO and DEA-treated ITO substrates were spin-coated with the perovskite precursor at 4000 rpm for 25 s to form the perovskite layers. Eighteen seconds before the end of the spin-coating process, 0.8 mL of diethyl ether was dripped on the perovskite surface. The resulting perovskite layers were heated for 15 min at 130 °C before naturally cooling to ambient temperature. Subsequently, both perovskite layers were spin-coated with the Spiro-OMeTAD solution for 30 s at 4000 rpm. The devices were finished by evaporating 60 nm Au electrodes onto the ITO/perovskite/spiro-OMeTAD and ITO/DEA/perovskite/spiro-OMeTAD.−9 Torr vacuum with a monochromatic Al-Kα X-ray source was used to collect the XPS spectra. A scanning electron microscope  and an atomic force microscope  AFM-5500) were utilized to study the morphologies. The perovskite’s crystallinity was examined using an X-ray diffractometer (Bruker D8 Advance (Bruker AXS Inc.), Cu-Kα radiation of 0.15406 nm). The UPS spectra were obtained using the ESCALAB 250Xi Contact angle images were obtained with an Ossila Contact Angle Goniometer. The FT-IR spectra were collected by using a Jasco  FT-IR spectrometer. Using a UV-Vis spectrophotometer (UV-2600), the absorption spectrum was measured. The steady-state PL spectra of the produced samples were looked at utilizing an Edinburgh PLS 980. Utilizing a transient state spectrophotometer (Edinburgh Institute F900) and a 485 nm laser, the TRPL decay of the perovskite layers was monitored. Under AM 1.5 G illumination with a power intensity of 100 mW cm−2, the J-V characteristic curves were measured with a source meter  2400) utilizing forward (−0.1 to 1.2 V) or reverse (1.2 to −0.1 V) scans from a solar simulator (XES-301S+EL-100). The delay time was set to 10 ms, and the step voltage was set to 12 mV. The EQE was calculated using QE-R systems . The EIS measurement was carried out using an electrochemical workstation (Zahner Zennium). The EIS data were fitted with help of ZSim-software version 3.20 with equivalent circuit parameters of R1 = 0.116, C = 0.00001 and R2 = 54.1 (for ITO-based device), and R1 = 0.116, C = 0.000001 and R2 = 29.6 (for DEA-treated ITO-based device).A Thermo Fisher Scientific  ESCALAB 250Xi under 10The cleaned ITO substrates were first spin-coated with DEA at 2000 rpm for 30 s, then dried at 100 °C for 10 min. X-ray photoelectron spectroscopy (XPS) was carried out to investigate the effect of DEA treatment on the surface chemistry of ITO. The XPS survey spectra of bare ITO and DEA-treated ITO are presented in To investigate the impact of DEA coating on the surface morphology of ITO, scanning electron microscopy (SEM) was conducted on bare ITO and DEA-treated ITO. cutoffE (secondary electron edges) and onsetE (Fermi edges), the intercepts of the tangents of the peaks with the extrapolated baselines were used. By estimating the difference between the incident photon energy  and cutoffE, the Fermi-level (FE) values of the bare and DEA-treated ITO were quantitatively determined. The UPS show an cutoffE of 16.64 eV and 17.21 eV for bare ITO and ITO/DEA, respectively. The EF values of bare ITO and ITO/DEA were estimated to be −4.58 and −4.01 eV, respectively, using the following equation.In order to get highly efficient PSCs in ESL-free architectures, one of the main obstacles is the energy-level mismatch between ITO and the perovskite, so it is essential to investigate how DEA affects the energy levels of ITO. FE of the ITO was significantly shifted by −0.57 eV after DEA treatment, which would be advantageous for the cathode’s ability to collect electrons. This is because, following DEA treatment, a favorable dipole moment formed on the surface of the ITO. As stated in previous studies, Pb-based compounds interact with –OH groups, while the amine groups in DEA tend to link with the ITO surface [The  surface ,35. As c2+ [2+ can improve the penetration of PbI2 into a TiO2 mesoporous layer [2 powder, we were able to further test the possible interaction between DEA and PbI2. Intriguingly, adding PbI2 to DEA has significantly altered the appearance, likely creating new products. As seen in 2 powder were combined to create an entirely distinct product that was white in color. The white mixture turns into a colorless liquid after heating  characteristic curves, and photovoltaic parameters of the as-prepared PSCs are depicted in ocV) of 1.096 V, a short-circuit current density (scJ) of 22.94 mA cm−2, a fill factor (FF) of 74.19%, and a power conversion efficiency (PCE) of 18.65% under reverse scan condition. In comparison, the DEA-modified PSC shows improved performance of up to PCE of 20.77%, a ocV of 1.109 V, a scJ of 24.45 mA cm−2, and an FF of 76.60% under the same scan condition. It is noteworthy that the DEA-modified PSC exhibits significantly better performance and less hysteresis in the J-V curves. The better layer quality and improved charge-carrier transport at the ITO/DEA/perovskite interface might be primarily responsible for the improved performance and decreased hysteresis, which is consistent with the dark J-V curves, as discussed below, where we observed a decrease in the density of trap states in the DEA-modified PSCs.We fabricated PSCs with and without DEA for the evaluation of photovoltaic parameters after carefully examining the morphology and substantial role of the DEA at the ITO/perovskite interface. The schematic diagram of the as-prepared device and energy levels of each layer are depicted in J-V measurement was carried out. As seen in trapN) can be determined with the equationTFLV, e, and L are the vacuum permittivity, relative dielectric constant, trap-field limit voltage, elementary charge, and perovskite layer thickness, respectively. Since trapN and TFLV are linearly related, a device with a smaller TFLV will exhibit fewer defects overall. As seen in TFLV (0.25 V) than the device with bare ITO (0.33 V), indicating DEA successfully reduces the defect density. The smooth morphology with fewer grain boundaries and the high-quality crystallinity of the perovskite layer deposited on the ITO/DEA substrate are responsible for the reduced trap density. Electrochemical impedance spectroscopy (EIS) measurements were conducted in order to learn more about how DEA affects the interfacial charge transport and recombination processes in the PSCs. ctR), which originates from the substrate and perovskite contact. Because ITO/DEA made better contact with the perovskite surface, the DEA-treated PSC showed a lower ctR value (29.76 kΩ) than the ITO-based device (ctR of 54.06 kΩ), indicating more efficient electron transfer and electron extraction at the corresponding interface. Additionally, measurements of external quantum efficiency (EQE) were made in order to assess how well the J-V and EQE curves agreed. The matching scJ values integrated from the EQE curves  [To further confirm the charge-carrier dynamics and charge-carrier recombination in the devices with bare ITO and ITO/DEA, the dark E curves d for botructure) ,46,47. MFurthermore, we acquired performance data from 9 PSCs with bare ITO and ITO/DEA, respectively, to show the reproducibility of the devices. The ITO/DEA-based PSCs exhibit improved performance and reproducibility when compared to PSCs with bare ITO, as shown in We used DEA to generate Sn-N and –OH–Pb chemical linkages between the perovskite layer and the ITO substrate. The interface contact, perovskite crystallinity, and film quality have all increased due to these chemical interactions, and the perovskite layer’s grain boundaries and trap states have decreased. Based on the creation of interfacial dipole layers, our results imply that the addition of DEA can modify the band misfit at the ITO/perovskite contact, hence reducing the voltage deficits. Transient PL, TRPL, and IS measurements revealed that the rates of electron extraction and charge transfer have dramatically improved. The PCE of the PSCs has significantly increased as a result, rising from about 18.65% to 20.77%. Additionally, DEA modification has increased the stability of the PSCs, with 80% PCE retention even after 550 h without encapsulation. This is likely because the DEA modification improved the interfacial contact and thin-film quality of the perovskite. Our research opens up scientific pathways for the facile fabrication of ESL-free PSCs.


T2 mapping of the liver is a potential diagnostic tool, but conventional techniques are difficult to perform in clinical practice due to long scan time. We aimed to evaluate the accuracy of a prototype radial turbo-spin-echo (rTSE) sequence, optimized for multi-slice T2 mapping in the abdomen during one breath-hold at 3 T.2 and 80 subjects  were investigated. A radial turbo-spin-echo (rTSE) sequence with and without fat suppression, a Cartesian turbo-spin-echo (Cart-TSE) sequence, and a single-voxel multi-echo STEAM spectroscopy (HISTO) were performed in phantom, and fat-suppressed rTSE and HISTO sequences were performed in in vivo measurements. Two approaches were used to sample T2 values: manually selected circular ROIs and whole liver analysis with Gaussian mixture models (GMM).A multi-sample (fat: 0–35%) agarose phantom doped with MnClR2 = 0.988) and with HISTO-T2s of water (R2 = 0.972) in phantom with an offset between rTSE and Cart-TSE maps (mean difference = 3.17 ± 1.18 ms). The application of fat suppression decreased T2 values, and the effect was directly proportional to the amount of fat. Measurements in patients yielded a linear relationship between rTSE- and HISTO-T2s .The rTSE-T2s values exhibited a strong correlation with Cart-TSE-T2s (The fat-suppressed rTSE sequence allows for fast and accurate determination of T2 values of the liver, and appears to be suitable for further large cohort studies.Radial turbo-spin-echo T2 mapping performs comparably to Cartesian TSE-T2 mapping, but an offset in values is observed in phantom measurements.•Fat-suppressed radial turbo-spin-echo T2 mapping is consistent with T2 of water as assessed by MRS in phantom measurements.•Fat-suppressed radial turbo-spin-echo sequence allows fast T2 mapping of the liver in a single breath-hold and is correlated with MRS-based T2 of water.• Magnetic resonance (MR) has been used to non-invasively diagnose and characterize different pathological conditions of the liver. By assessing MR parameters, conditions such as steatosis , iron ovTo increase patient compliance and clinical throughput, the assessment of all parameters needs to be fast. Desired elimination of breathing artifacts in the abdominal examinations underlines the need for optimized accelerated scan protocols. Whereas most of the methods from the multi-parametric protocols are fast enough , the data acquisition for the assessment of tissue-specific T2 relaxation times with the use of conventional multi-spin-echo techniques  is consiIn animal models, T2 mapping was suggested to be a marker of hepatic fibrosis , 11, witIt has been proposed that long acquisition times and sensitivity to body motion of conventional T2 mapping can be overcome by radially sampled techniques combined with turbo-spin-echo (TSE) imaging and a tiered reconstruction . Radial Among fibrosis and iron overload, fat accumulation is another pathology involving liver diffusely. It has been shown that fat is a confounder in T2 measurement by direct comparison of fat-suppressed and non-fat-suppressed multi-echo T2 acquisitions in iron-overloaded patients . While tTherefore, the goal of this study was to evaluate a fat-suppressed radial TSE (rTSE) sequence for accuracy of T2 mapping and feasibility in clinical protocol. The parameters were optimized for measurement in the liver at 3 T during single breath-hold, while keeping sufficient in-plane resolution and volume coverage (number of slices = 5). MRS-based T2 measurement taken as quantitative gold standard in both in vitro and in vivo conditions was complemented by Cartesian T2 mapping in phantom measurements.(eff) times. The radial sequence used here generates individual TE(eff) images via echo-sharing. The general idea of echo-sharing is to use only the views with given TE in the center of the k-space, which determines the image contrast, and views at other TEs are included in the outer part of the k-space. The inclusion of the views with other TEs can be done in several ways; in this study, a “full-tier” method  was used [T2 relaxation times are being calculated by exponential fit to multiple images with different TEwas used .To reduce the streaking artifacts arising from T2 decay and object motion, schemes that distribute the views from different TR periods were proposed . Here, aThe sequence was first tested in phantoms for its accuracy, and the effect of fat suppression on the T2 values was investigated. Finally, the sequence was evaluated in a cohort of patients and volunteers under clinical settings.2 (in concentration 0.28–0.48mM) to mimic human liver in vivo T2 values  with peanut oil (0–35%) and lecithin as emulsifiers, doped with MnCl4 ± 4 ms ). All saDatasets of 78 patients undergoing abdomen MR examination between October 2018 and June 2019 were selected for the study  equipped with a 64-channel body coil and a 32-channel spine coil.All measurements were performed on a 3-T MR system   allowingThe fat-suppressed rTSE-T2 map was integrated into the multi-parametric abdomen protocol targeted at patients undergoing abdomen examination at our clinic. The data for 5 slices of the T2 map were acquired across the largest partition of the liver during a 17-s breath-hold. The protocol included HISTO sequence as well, serving for FF and water T2 assessment, which were evaluated inline. The spectroscopic voxel was placed in the liver parenchyma avoiding large vessels, biliary tracts, and liver boundaries.n = 7), the examination was repeated on the same examination date and the subject was taken out of the magnet and asked to sit up, and the coil was unplugged between the measurements.For the evaluation of T2 mapping variability in test-retest measurements on healthy volunteers  analysis by a single operator with 2 years of experience in liver mapping. In the phantoms, circular ROIs as large as the sample permitted, excluding the borders, were placed on the slice through the middle of the sample. In vivo liver data were evaluated with two approaches. First, the simpler approach  was to draw two to three circular ROIs placed in the liver parenchyma, excluding large vessels, ducts, and streaking artifacts, and to calculate T2 as the mean of the ROI values. In the second approach , the slice with the largest liver cross-sectional coverage was selected, and a free-hand ROI encompassing the whole liver was drawn manually. Since the ROI included sections other than pure liver parenchyma, like vessels and lesions, the ROI T2 values were fit to a Gaussian mixture model implementing the expectation-maximization (EM) algorithm. The Gaussian mixture model (GMM) is a soft-clustering method, which allows a pixel to belong to more than one type of tissue with a certain probability , 22. TheR square was reported. Bland-Altman analysis was performed between radial and Cartesian acquisition in phantom, in test-retest study, and between the ROI and GMM approach. One-sample t-test on the differences against zero was carried out to test for statistical difference. For test-retest study, coefficients of variation (CoV) were calculated. Statistical analysis was performed in SPSS 24.0 and p < 0.05 was considered statistically significant.Data were presented as counts for categorical variables and as mean ± standard deviation (SD) for normally distributed variables and as median ± interquartile range (IQR) for non-normally distributed continuous variables. For the comparison of the methods and approaches, regression analysis was performed and adjusted R2 = 0.972, p < 0.001, Fig. R2 = 0.988, p < 0.001, Fig. p < 0.001).Table R2 = 0.273, p = 0.165 and R2 = 0.170, p = 0.490, respectively).The HISTO FFs of the samples ranged from 0.1 to 36.0% Table . The fatThe exclusion criteria for the in vivo analysis were severe streaking artifacts (4) and inadequate liver coverage with rTSE 1), leaving 73 patients to evaluate. Clinical characteristics of patients and summary of primary indications for MR exams are given in Table , leavingR2 = 0.943 (p < 0.001) for ROI analysis and R2 = 0.831 (p = 0.003) for GMM analysis with small differences that were not statistically significantly different from zero  with a slope of 1.12 (p < 0.001) and intercept 7.49 ms (p = 0.32) for ROI analysis and with a slope of 1.01 (p < 0.001) and intercept 10.16 ms (p < 0.001) for GMM analysis  and were in a good agreement with mean difference of 0.45 ms that was not statistically significant (p = 0.079), and upper and lower limits of agreement 4.87 and − 3.97 . Note that the analyzed range of R2 (1/T2) values was much broader than in our study, which might be the source of stronger correlation compared to our study  for the iron assessment in the liver . Regressnding to  vs ROI/GSome authors studied liver T2s as a marker of disease using only dual-echo TSE sequences, which introduces lower statistical certainty in the fitting algorithm . DespiteAs the limitations of our study, we should certainly name: (i) missing of direct comparison to a “gold standard” for in vivo liver T2 mapping, and it is yet unclear which method that could be. The theoretical equivalence of spectroscopy (HISTO) and imaging has not been established yet, and has to be further investigated . (ii) ThIndirect (stimulated) echoes can confound T2 values, and approaches to correct for this have been proposed, also for estimation of T2s from highly under-sampled data . This coIn conclusion, the radial T2 mapping with short acquisition times, thus enabling breath-hold acquisition, presented here compares favorably to other methods both in phantoms and in vivo, works reliably in a clinical protocol, and appears to be suitable for further studies of diffuse liver disorders and to be a part of multi-parametric protocols.


Shigella sonnei remains the second most common cause of shigellosis in young children and is now increasingly dominant across developing countries. The global emergence of drug resistance has become a main burden in the treatment of S. sonnei infections and β-lactam antibiotics, such as pivmecillinam and ceftriaxone, are recommended to be used as second-line treatment. They work by inhibiting the biosynthesis of the peptidoglycan layer of bacterial cell walls, in which the final transpeptidation step is facilitated by penicillin-binding proteins (PBPs). In this study, using protein homology modelling, we modelled the structure of PBP6 from S. sonnei and comprehensively examined the molecular interactions between PBP6 and its pentapeptide substrate and two antibiotic inhibitors. The docked complex of S. sonnei PBP6 with pentapeptides showed that the substrate bound to the active site groove of the DD-carboxypeptidase domain, via hydrogen bonding interactions with the residues S79, V80, Q101, G144, D146 and R240, in close proximity to the catalytic nucleophile S36 for the nucleophilic attack. Two residues, R240 and T208, were found to be important in ligand recognition and binding, where they formed strong hydrogen bonds with the substrate and β-lactams, respectively. Our results provide valuable information on the molecular interactions essential for ligand recognition and catalysis by PBP6. Understanding these interactions will be helpful in the development of effective drugs to treat S. sonnei infections. Shigella sonnei is one of the four causative agents of shigellosis, an intestinal infection that is also known as bacillary dysentery. The species is considered as the second most common cause of diarrhea in young children in rapidly industrializing countries and together with Shigella flexneri, the species is responsible for more than 90% of the global shigellosis cases ..S. sonneGiven the importance of the β-lactam antibiotics, such as pivmecillinam and ceftriaxone, in the treatment of shigellosis, as recommended by the WHO as the second-line treatment in all age groups , we sougHowever, unlike ceftriaxone—which was predicted to shield the catalytic site but did not interact the catalytic nucleophile S36—pivmecillinam had direct contact with S36 via the carbonyl group of its penam b. This iE. coli PBP6, S40 has been shown to form a hydrogen bond with the penam ring of ampicillin in the acyl–enzyme complex, but relative to the pre-acylation complex with the pentapeptide substrate  with the carbonyl oxygen atom of the penam ring of ampicillin, but compared to pivmecillinam, ampicillin had only two hydrogen bonding contacts with PBP6. The other contact was a strong 1.8-Å hydrogen bond with T208 c. Albeitubstrate b, there ubstrate d.S. sonnei PBP6 and its substrates would allow us to understand the molecular interactions and conformational changes, important for ligand recognition and catalysis by the enzyme. Together with the previous studies on other D-Ala-D-Ala carboxypeptidases, such as the E. coli PBP5 [E. coli PBP6 [S. sonnei infections. S. sonnei has been continuously becoming resistant to antibiotics and shows an extraordinary ability to acquire antimicrobial resistance genes from other pathogenic bacterial species, such as Klebsiella spp. and E. coli [Shigella spp. For instance, a previous study has focused on genetic characterization and antimicrobial resistance of Shigella spp., where ESBL genes, such as blaTEM-1, blaCTX-M, blaOXA-1, and blaSHV-12, are found to be dominant in the S. sonnei isolates. Resistance to ampicillin is the most common, in which 97.7% of the total 474 Shigella isolates are resistant to the broad-spectrum antibiotic, whereas 26.0% of the isolates are resistant to ceftriaxone, the third-generation cephalosporin [Shigella isolates, with 53.7% of the isolates carrying ESBL genes [Shigella spp. that confer the resistance to β-lactam antibiotics, such as pivmecillinam and ceftriaxone, which are currently used as the second-line treatment against shigellosis [S. sonnei PBP6 and its substrate, as well as antibiotic inhibitors, have not been structurally evaluated. As the high-resolution 1.80-Å crystal structure of the E. coli PBP6 in complex with the pentapeptide substrate has been published [S. sonnei PBP6 are warranted. Shigella spp. and E. coli are closely related and genetically similar species [S. sonnei PBP6 shares 99% sequence identity with PBP6 from E. coli (S. sonnei and comprehensively examined the molecular interactions between PBP6 and its substrate and inhibitors.Elucidating the complex structures between the oli PBP5  and the oli PBP6 , they pr E. coli ,4. In thlosporin . AnotherBL genes . This is species . For exa E. coli . Thus, uX-X-K, S-X-N and K-T-G. The S-X-X-K motif is the site of acylation by both the peptide substrate and β-lactams, and is important for the substrate binding and catalysis [D-Ala-D-Ala carboxypeptidases, as is the case with PBP6, cleave the peptide bond between the two terminal D-alanines of the pentapeptide stem during the cross-linking of the peptidoglycan polymers, creating a mesh-like structure . Like otatalysis ,11. The atalysis . Structuatalysis . Mutatioatalysis .E. coli PBP6 [S. sonnei PBP6 complexes with the pentapeptide substrate, as well as the two β-lactam antibiotics, provide structural context to understand this mimicry and additionally give insights into the inhibition mechanism of β-lactams. Our structures (It is widely accepted that nucleophilic attack of the β-lactam carbonyl by the catalytic residue serine of transpeptidases causes the opening of the β-lactam ring and the formation of a stable and irreversible acyl–enzyme complex ,10. Howeoli PBP6 , our S. ructures  revealedructures a. The β-S. sonnei PBP6 and its ligands not only provides information on their intermolecular interactions but can also be utilized to develop high-affinity and specialized PBP6 inhibitors. Like other PBPs, PBP6 recognizes β-lactams as they mimic the D-Ala-D-Ala moiety. From our molecular docking simulations, we found that the pentapeptide substrate and the β-lactam antibiotics can fit into the active site groove of the DD-carboxypeptidase domain, in close proximity to the catalytic nucleophile S36 for the nucleophilic attack. Two residues, T208 and R240, were found to be important in ligand recognition, where they made strong hydrogen bonds with β-lactams and pentapeptides, respectively. Our current computational analysis provides valuable information on the molecular interactions essential for ligand recognition and catalysis by PBP6. Understanding these interactions will be helpful in the development of new effective drugs to treat S. sonnei infections. Further in vitro studies using the S. sonnei PBP6 are warranted to ensure the specificity of the intermolecular interactions between the protein and its ligands.Elucidating the complex structures between the


The inter-rater reliability of the Thai-HFHAT-SF was 0.82 (95% CI: 0.71–0.89). The test–retest reliability was 0.77 (95% CI: 0.60–0.87) for the older person group, 0.85 (95% CI: 0.73–0.91) for the caregiver group, and 0.60 (95% CI: 0.29–0.77) for the VHV group. The new 28-item scale focused on home fall hazards and can be conducted in 10–15 min. Thai-HFHAT-SF is suitable for home hazards assessment among elderly in Thailand.The Thai-Home Fall Hazard Assessment Tool (Thai-HFHAT) was developed to identify the fall risk among the elderly arising from their home environment. However, it is more time consuming for large items. Therefore, this study developed a short-form of Thai-HFHAT (Thai-HFHAT-SF). In phase I, we developed the Thai-HFHAT-SF by performing a confirmatory factor analysis (CFA) of 450 rural elderly people. In phase II, a total of 105 participants; 50 elderly people, 50 caregivers, and 5 village health volunteers (VHV) were recruited to examine the reliability of the Thai-HFHAT-SF. Intra-class correlation coefficient (ICC) was used to analyze the inter-rater and test–retest reliability. Factor analysis selected 28 out of the 69 original Thai-HFHAT items in 4 components: indoor area, garage, outdoor areas, and risky spots/areas including pets. The factor loading was 0.67, 0.60, 0.32, and 0.31 in each component. The fitness index indicated that this model was fit ( Falls are a major public health problem in the elderly. The WHO Global Report on Falls Prevention in Older Age estimates that approximately 28–35% of adults aged over 65 years will report at least one fall each year . Thailanp = 0.0001) [The environmental factors include home and environmental hazards, such as insufficient lighting, uneven flooring, and loose mats. Steep or narrow stairs/steps were also important factors that caused falls in the elderly . Falls c 0.0001) . HoweverIn Thailand, the Thai Home Falls Hazards Assessment Tool (Thai-HFHAT) has been developed and accepted as the only reliable home hazard fall risk screening instrument. This tool has 69 questions and takes about 45 min to administer. It has been designed in such a way that the elderly can fill it out with or without help from Thai village health volunteers (VHV). The ICC of inter-rater reliability and test–retest reliability for the Thai-HFHAT was 0.87 (95% CI: 0.78–0.93) and 0.78 (95% CI: 0.58–0.89), respectively [This cross-sectional study was conducted at Nakhon Si Thammarat, Thailand from 26 June 2021, to 15 August 2021, and consisted of 2 phases. A confirmatory factor analysis (CFA) of the 69-question Thai-HFHAT was performed in phase I. A short-form of the Thai-HFHAT (Thai-HFHAT-SF) based on the original version was developed, and reliabilities of the Thai-HFHAT-SF were evaluated in phase II.The survey method examined the achievement of fall risk screening data of 450 elderly  who wereThe cross-sectional survey was designed to investigate the content validity and the inter-rater reliability of the Thai-HFHAT-SF. The test–retest reliability was evaluated using a prospective design that all participants answered (Thai-HFHAT-SF) and 2 weeks later, the Thai-HFHAT-SF questions were repeated. This study was approved by the Institute Review Board Walailak University (IRB reference no. WUEC-20-302-01) before commencement. This phase was conducted in the elderly, caregivers, and village health volunteers (VHV) recruited from Tha Sala District, Naknon Si Thammarat by convenience sampling method. The inclusion criteria of the elderly were both genders and aged ≥ 60 years with fluency in Thai who were willing to participate in this study. In addition, caregivers aged < 60 years (20–50 years) who spent most of their time taking good care of the elderly and VHV were also included in this study. The elderly people who were unable to perform daily activities and those who exhibited the symptoms of early-onset dementia were excluded. A number of 50 elderly were included and eligible for the evaluation of inter-rater and test–retest reliabilities . In thisThe procedure of test–retest reliability of the Thai-HFHAT-SF was performed twice by the elderly during the first home visit and 2 weeks after the first visit, as recommended by Marx et al. . MoreoveData were analyzed using the statistical package for the social sciences (SPSS), version 22.0 for Windows . Confirmatory factor analysis for categorical data  was also performed.p < 0.001), (ii) normality determined from skewness (≤2) and kurtosis (≤7), (iii) suitability of data determined from Kaiser–Meyer–Olkin: KMO must be close to 1, and (iv) correlation determined from chi-square must be (p < 0.05). The secondary order CFA was performed based on the correlation matrix to evaluate the construct validity of the model with 9 components. Maximum likelihood estimation (ML) was conducted to determine values for the parameters in the examination of construct validity. Structural equation modeling (SEM) was used to determine the fitness of the model using the following statistics: Chi-square (χ2), chi-square to df ratio (χ2/df), GFI, AGFI, CFI, SRMR, and RMSEA. A comparison of component weights and empirical data was carried out to determine the weight of four factor components.Demographic data were analyzed using descriptive statistics. The assumptions of factor analysis were to meet the criteria of: (i) univariate outlier standardized scores must be greater than or equal to 3.30 (|z| ≥ 3.30) and multivariate outlier determined from the Mahalanobis distance value must be  and ICC, were used to evaluate inter-rater and test–retest reliabilities of the Thai-HFHAT-SF, respectively. Reliability results were classified into 4 levels: an ICC < 0.50 was classified as having poor reliability, 0.50–0.74 as moderate, 0.75–0.90 as good, and >0.90 as excellent reliability [The content validity index (CVI) was classified as follows: 1.00 was classified as not relevant, 2.00 as somewhat relevant, 3.00 as quite relevant, and 4.00 as relevant . The iteiability .The CFA of the 69-question Thai-HFHAT was performed. The results of the analysis were categorized into two subsections: the results of assumption tests and the results of CFA.p < 0.001). Results showed that no study subjects exhibited a z score of ≥3.30 as an outlier, but 8 of the total subjects had Mahalanobis distance (p < 0.001). We determined to continue our study with a total number of 442 study subjects. Data distribution of each question listed in 9 sections of the Thai-HFHAT was examined and a normal distribution was indicated by the graph depicting a bell curve. Maximum likelihood estimation was conducted in the confirmatory factor analysis based on skewness of 2 or less and kurtosis of 7 or less [p < 0.05). Results showed that χ2 was 466.54 (p < 0.001), indicating that the set of data could be used for confirmatory factor analysis.Univariate outlier and multivariate outlier tests were performed to prevent misinterpretation of the results. Univariate outlier values were used to calculate standardized scores (|z| ≥ 3.30), whereas multivariate outlier values were used for finding Mahalanobis distance  was observed. Weights were as follows: 0.67, 0.60, 0.32, and 0.31 with factor loadings ranging from 0.10 to 0.71. The fitness index showed values of  was fit .The demographic characteristics of 150 subjects are shown in Twenty-two out of the 29 items on the Thai-HFHAT had excellent agreement (I-CVI = 1) and 7 items had acceptable agreement (I-CVI = 0.7). The content validity for the scale of the instrument was good (S-CVI = 0.9). The value of I-CVI and S-CVI are shown in The ICC of inter-rater reliability for the Thai-HFHAT-SF was 0.82 (95% CI: 0.71–0.89). The mean (±SD) scores by the elderly, caregiver, and VHV groups were 4.56 (±3.29), 4.23 (±3.22), and 3.35 (±2.65), respectively, as shown in The main purpose of this study was to develop the Thai-HFHAT-SF. The results showed that the tool was reduced to 28 questions in four factor groups. Therefore, the Thai-HFHAT-SF was shorter, took less time, and was more precise when compared to the original version (69-question Thai-HFHAT)  and consThe item content validity of the Thai-HFHAT-SF was evaluated and 90% of the instrument’s question items had an excellent agreement (I-CVI = 1.00). However, 10% had an acceptable agreement (I-CVI = 0.67) because the opinion of an architectural expert showed that the definition of home hazards represented the risk to overall health, not merely hazards that cause the falls. For example, the item “There is no bright light for activities in the garage” was rated a score of 2 with the reasoning that more items should be added to cover the size of the garage’s area that could cause injuries to the elderly while getting in/out of the car. Nevertheless, the content validity for the scale of the Thai-HFHT-SF was good .The inter-rater reliability for the Thai-HFHAT-SF was 0.82. The findings were consistent with the previous study that examined the inter-rater reliability of the 69-question Thai-HFHAT, showing ICC = 0.87  because The mean score of the Thai-HFHAT-SF was highest in the elderly group. This was due to most of the elderly having health issues with falling being one of the health issues posing a serious threat to their health ,23, causThe ICC of the test–retest reliability was 0.77, which was consistent with the previous study conducted with the 69-question Thai-HFHAT with an ICC of test–retest reliability of 0.78  and RomlThe means scores for the Thai-HFHAT-SF by the elderly group, the caregiver group, and the VHV group were slightly different between the 1st and 2nd home visits. This may contribute to the environment in the homes of the elderly or behavior changes of the elderly after the first home visit. As the elderly completed the first-time rating of the tool, they may pay more attention to their surroundings and make some changes to the environment to make their home safer . This phThe question items of the Thai-HFHAT-SF were reduced to 28. The repetitive questions about lighting, slippery floors, or uneven ground were found when assessing each room in the Thai-HFHAT version. The findings in this study also confirmed that the 28-question Thai-HFHAT-SF had good inter-rater reliability, making this instrument acceptable as a reliable fall risk instrument for Thais. We recommend the Thai-HFHAT-SF for environmental assessment; however, fall prevention should cover both environmental and biological assessment. Kim et al. reported that the new fall risk assessment (FRA) system may be used to evaluate the biological hazards because of its moderate to excellent inter-rater reliability and sensitivity to change in community-dwelling older adults . The limThe inter-rater reliabilities of the HOME FAST-SR and Thai-HFHAT were moderate and good, respectively. The test–retest reliability of the 28-question Thai-HFHAT-SF was good. The instrument appears to be a reliable instrument for identifying home hazards that pose an increased risk for falls, and for screening the elderly who have potential risk for falls in the study population. We suggest that prospective studies are needed among various populations to ensure that this instrument can be applied to the majority of the elderly in Thailand and used for fall prediction in all types of Thai homes.


S. aureus (ATCC 25923). The characterization of NPs was analyzed using scanning electron microscopy (SEM), Zetasizer and Fourier-transform infrared spectroscopy (FTIR). The drug encapsulation efficiency and loading capacity percentages of NPs were calculated by the spectrophotometric method. The drug release of N-loaded PVA-R-PLGA NPs was determined by the dialysis bag method. The antibacterial and antibiofilm activity of N-PVA-R-PLGA NPs was determined. PVA-R-PLGA-NPs were found to be spherical with sizes of ~140 nm, according to the SEM analysis and surface charge of N-PVA-R-PLGA NPs −53.23 ± 0.42 mV. The sustained release of N (≥72% after 6 h) was measured in PVA-R-PLGA-NPs. The encapsulation efficiency percentage of N-PVA-R-PLGA NP was 78%. The MIC values of free nisin and N-PVA-R-PLGA NPs were 256 μg/mL and 64 μg/mL, respectively. The antibiofilm inhibition percentages of free nisin and N-PVA-R-PLGA NPs were 28% and 72%, respectively. These results reveal that N-PVA-R-PLGA NPs are a promising formulation for use in infections caused by S. aureus compared to free nisin.In this article, nisin(N)-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) were prepared using the single-solvent evaporation method with a rhamnolipid(R) cosurfactant. The antibacterial–antibiofilm effects of the prepared formulation and free nisin were evaluated against  Staphylococcus aureus (S. aureus) is considered as a pathogen that threatens human health by the World Health Organization. S. aureus attaches to medical implants and tissues. After this attachment, bacterial cells form a polymer-based matrix layer, forming structures called biofilms [biofilms ,2. Previbiofilms . To combbiofilms . Also, ubiofilms ,6,7. Accbiofilms . Accordibiofilms . These rLactococcus lactis strains. This antimicrobial polypeptide consists of 34 amino acids and has a molecular weight of 3.5 kDa. Nisin causes the effective inhibition of Bacillus and Clostridium spores as well as a broad spectrum of Gram-positive bacteria (Staphylococcus and Listeria) [Listeria strains encapsulated in phosphatidylcholine (PC) liposomes containing chitosan or chondroitin sulfate by thin-film hydration method were evaluated. It was determined that encapsulated nisin has 21% more efficacy in inhibiting Listeria monocytogenes than nisin alone [Lactobacillus sakei was determined under different conditions and evaluated in terms of drug release profile. It showed a strong inhibitory activity by giving a value of 2.70 log CFU/mL at pH 4.5 and showed sustained release activity for 2 weeks in an 8-month drug release profile [The drug encapsulated in our study is the nisin protein, an antimicrobial polypeptide (AMP). Nisin, a bacteriocin, is a cationic and amphiphilic inexpensive AMP produced by isteria) . Nisin iisteria) . In a stin alone . Accordi profile . In a st profile . The addWe aim to investigate the effect of PLGA NPs for nisin, prepared using rhamnolipids, which have demonstrated their effective properties in our previous study . Nisin-lStaphylococcus aureus (ATCC 25923) was used as the reference pathogen and was maintained in Mueller Hinton Agar (MHA) at 37 °C.Nisin 2.5%, PLGA , 75:25, PVA , acetone (HPLC grade), ethanol (100%) and dialysis bag (MWCO: 12 kDa) were purchased from Sigma Aldrich. Rhamnolipid  was purchased from AGAE Technologies. NutriCulture Phosphate-Buffered Saline (PBS) was purchased from EcoTech Biotechnology. Before preparing the drug delivery system, nisin (2.5%) was purified by ethanol extraction. A total of 200 mg of commercially purchased nisin (2.5%) was weighed and suspended in 10 mL of 50% ethanol. The suspension was then stirred at room temperature for 8 h and then centrifuged at 1.520 g for 5 min. After centrifugation, the supernatant was lyophilized. The nisin protein obtained after lyophilization was dissolved with 0.02 N HCL and used in the drug delivery system . The druA zeta sizer Nano ZS  was used for the determination of Mean particle size (d.nm), polydispersity index (PDI) and zeta-potential values NPs. In brief, the measurements were determined by the dynamic light scattering method at 20 °C, and samples for analysis were prepared with appropriate dilutions.For morphological characterization, NP formulations were coated with gold and examined under SEM (FEI Quanta Feg-450). The magnification of the image was set to ×20,000 .−1 [Infrared spectra will be obtained by using NP formulations and the nisin protein FT-IR to determine the polymer-active substance interactions and to detect the drug presence in the prepared drug delivery formulations. The IR (infrared) spectra of the samples will be acquired in a reflectance (ATR) mode and the spectral region of 400 to 4000 cm−1 .The solution in the release medium after dialysis of excess nisin from the NPs was measured to determine the EE% and DL% of the NPS. Then, the solution was measured with Multiskan™ GO Microplate Spectrophotometer at 215 nm, and the amount of N was quantified using an N calibration curve. EE% and DL% were measured using the following equations ,20. The In vitro release test of Üstün and Örtücü (2022) was carried out by making some changes to the protocol according to the dialysis bag technique . For thiNP formulation was measured spectrophotometrically at 215 nm at specific time intervals .NPs’ formulations (N-PLGA-NPs and the PLGA-NPs) were dissolved in a sterile buffer solution in the same concentration. The liquid dilution method was used to measure the MIC (minimum inhibitory concentration) value to determine antibacterial activity. In summary, 100 µL of bacterial cells at 0.5 McFarland concentration and specific concentrations of substances  were used in 96-well plates. The total volume was made up to 200 µL. Medium containing only medium was used as a negative control, and the bacterial cell group was used as the positive control. MIC values of test samples were recorded as the lowest concentration that inhibited bacterial growth after 24 h of incubation at 37 °C .S. aureus) adjusted to 0.5 McFarland were added to 96-well plates with polystyrene properties at 37 °C and incubated for 48 h. At the end of the period, all the media contents were removed, and all wells were washed three times with PBS. The wells were then treated with 0.5% crystal violet, and after washing, the biofilm mass was suspended with 30% acetic acid. The biofilm% inhibition was measured by using the following equations [Antibiofilm activity of nisin and NP formulation were determined with the crystal violet (CV) assay. For the biofilm formation inhibition test, 50 µL of NP formulations (in the same concentrations and dissolved in PBS), 75 µL of MHB medium, and 75 µL of 1-day-old bacterial cells (quations . Untreatp < 0.05) by one-way ANOVA .The results were determined as mean ± SD and analyzed for statistical significance  added rhamnolipids to chitosan NPs, causing the NPs to decrease in size and polydispersity index and show a more positive surface charge and improved stability. It was determined that the formulation prepared with the combination of rhamnolipids showed more effect against osan NPs . In the detected . In the detected .N-PVA-R-PLGA NPs had spherical morphology and zeta potential suitable for the nanobiotechnological drug delivery system in our study. The negative charge of NPs may be due to the surface charge of PLGA. The high zeta potential value provides stability to the nanocarrier system by preventing the aggregation of molecules. The high zeta potential of nisin-loaded PLGA NPs provides an advantage for the drug delivery system. For the homogeneous distribution of NPs, the PDI value should be between 0 and 1, and according to the results, N-PVA-R-PLGA NPs have a homogeneous distribution . The sizIn addition, in our previous study, PLGA NPs prepared with a PVA-R surfactant, compared to PLGA nanoparticles prepared only with a PVA surfactant, showed superior properties in terms of size, zeta potential and PDI .Nisin release from PLGA NPs showed biphasic release, burst phase and the second phase . An in vS. aureus compared to free N. (four times more). These results were found to contain similar results to those reported by da Silva et al. (2014) and Üstün and Örtücü (2022) [According to MIC values, N-PVA-R-PLGA NP was effective in reducing the bacterial viability of ü (2022) ,14. Theyü (2022) .Incorporating AMP of different natures  into nanobiotech drug delivery systems represents an effective therapeutic strategy to combat biofilms. NPs can be developed to increase penetration into cells and selectively target or release drugs. Moreover, the use of NPs as carriers allows an increase in drug efficacy by overcoming resistance mechanisms in bacteria. The antibiofilm inhibition percentage of N-PVA-R- PLGA NP was 72% .p < 0.0001)) shows that nisin encapsulation increases the antibiofilm efficiency approximately 2.5 times. According to the study of Niaz et al. (2019), rhamnosomes were made by combining nano-liposomes with rhamnolipids. An approximately 80% reduction in biofilm biomass was observed by loading nisin into rhamnosomes. In this study, the nisin protein was loaded with rhamnosome nano-vesicles, and its antibiofilm properties were investigated after treatment with crystal violet assay. According to the results, it was determined that the drug delivery system prepared with rhamnolipids reduced the attachment of biofilm cells by reducing the tension between the biofilm cells and the surface [The antibiofilm inhibition percentage of nisin is 28%, and the difference (significant difference (****  surface .Helicobacter pylori biofilm by eliminating EPS and also reducing both the adhesion and colonization of bacteria [In one study, an NP formulation containing rhamnolipids and phospholipids was used to encapsulate amoxicillin and pectin sulfate, an anti-adhesive agent. This study demonstrated that the nanocarrier could significantly disrupt the bacteria .In our previous study, we benefited from the slow release of the active ingredients of PLGA and the advantages of rhamnolipids. According to these studies, rhamnolipids appear as encapsulation agents or cosurfactants. The nisin delivery performed in this study with PLGA NPs may result in the development of a new therapeutic system.S. aureus threaten human health, and researchers have turned to nanobiotechnology for this in recent years. The use of nanobiotechnological drug delivery systems to increase the effectiveness of antimicrobial agents is very promising. For this purpose, PLGA NPs prepared with a PVA-R cosurfactant were used in our study and examined in terms of zeta potential, intermolecular interactions, PDI, size, in vitro release, drug encapsulation efficiency and antibiofilm activity. As a result, we found that nisin-loaded PLGA NPs are a more suitable drug delivery system compared to nisin alone.Biofilms originating from


The detection of coinfections is important to initiate appropriate antimicrobial therapy. Molecular diagnostic testing identifies pathogens at a greater rate than conventional microbiology. We assessed both bacterial coinfections identified via culture or the BioFire® FilmArray® Pneumonia Panel (FA-PNEU) in patients infected with SARS-CoV-2 in the ICU and the concordance between these techniques.This was a prospective study of patients with SARS-CoV-2 who were hospitalized for no more than 48 h and on mechanical ventilation for no longer than 24 h in 8 ICUs in Medellín, Colombia. We studied mini-bronchoalveolar lavage or endotracheal aspirate samples processed via conventional culture and the FA-PNEU. Coinfection was defined as the identification of a respiratory pathogen using the FA-PNEU or cultures. Serum samples of leukocytes, C-reactive protein, and procalcitonin were taken on the first day of intubation. We analyzed the empirical antibiotics and the changes in antibiotic management according to the results of the FA-PNEUM and cultures.Staphylococcus aureus (37.5%) and Streptococcus agalactiae (20%), and those detected by culture were Staphylococcus aureus (34.78%) and Klebsiella pneumoniae (26.08%). The overall concordance was 90.1%, and when stratified by microorganism, it was between 92.7 and 100%. The positive predictive value (PPV) was between 50 and 100% and were lower for Enterobacter cloacae and Staphylococcus aureus. The negative predictive value (NPV) was high (between 99.1 and 100%); MecA/C/MREJ had a specificity of 94.55% and an NPV of 100%. The inflammatory response tests showed no significant differences between patients whose samples were positive and negative for both techniques. Sixty-one patients (55.45%) received at least one dose of empirical antibiotics.Of 110 patients whose samples underwent both methods, FA-PNEU- and culture-positive samples comprised 24.54% versus 17.27%, respectively. Eighteen samples were positive in both techniques, 82 were negative, 1 was culture-positive with a negative FA-PNEU result, and 9 were FA-PNEU-positive with negative culture. The two bacteria most frequently detected by the FA-PNEU were The overall concordance was 90.1%, and it was between 92.7% and 100% when stratified by microorganisms. The positive predictive value was between 50 and 100%, with a very high NPV. Critically ill patients with COVID-19 have a high mortality rate 38.4%), mainly related to advanced age, the severity of illness on intensive care unit (ICU) admission, vasopressor support, and renal replacement therapy . In the 8.4%, maip < 0.001)  or endotracheal aspirate [ETA] for conventional culturing and the FA-PNEU. Of the total volume of samples, 5–10 ml (mL) was distributed for FA-PNEU testing, 5–10 mL for conventional culturing, and another 5–10 mL stored for lung microbiome analysis with the extraction of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) for metagenomic and metatranscriptomic sample sequencing. (The results of these tests are part of another study.) According to microorganism identification through the FA-PNEU, antibiotics were initiated, considering the pathogen and resistance profile; consequently, with bacterial culture results and antibiograms, definitive therapy was adjusted according to the treating physicians’ criteria.To describe whether the inflammatory response markers were associated with pulmonary coinfection, whole blood samples for leukocyte count and serum for C-reactive protein and procalcitonin were taken on the first day of intubation. We analyzed the empirical antibiotics after taking samples from the lower respiratory tract and the changes in antibiotic management according to the results of the FA-PNEUM and cultures.This study was conducted according to the regulations of the Universidad Pontificia Bolivariana ethics committee and the committees of each of the participating institutions. Informed consent was obtained from the patient or their legal representative for sample handling and accessing registered information in medical records.Testing for SARS-CoV-2 was performed by real-time polymerase chain reaction (RT–PCR) on nasopharyngeal swabs, following WHO and/or CDC protocol, in an Allplex SARS-CoV-2 assay  to amplify the SARS-CoV-2 E, RdRp/s, and N genes.2 for 2 days). Afterward, microorganisms that grew in significant amounts according to the guidelines of standard laboratory procedures were quantified (upon growth at or above 104 CFU/mL for a mini-BAL fluid sample or 105 CFU/mL for an ETA sample) and subcultured to isolate them if there were two or more microorganisms on nonselective plates. If they were negative, they were kept for further observation for up to 3 days. Cultures for viruses or atypical bacteria were not performed.Respiratory samples were subjected to Gram staining and conventional semiquantitative cultures. The samples were not processed when a quality level of 0 or 1 was detected by the Murray/Washington criteria, based on the number of squamous cells and neutrophils per field . Ten micCandida spp. and Enterococcus faecalis was considered negligible.Bacterial species-level identification was conducted using VITEK® 2 identification (ID) cards. Antimicrobial susceptibility testing (AST) was performed by VITEK® 2  following the manufacturer’s instructions. The tests were interpreted according to current Clinical & Laboratory Standards Institute (CLSI) guidelines . The preThe FA-PNEU is a syndromic panel based on multiplex PCR targeting 18 bacterial pathogens , nine viruses, and seven determinants of resistance . Viral, fungal, and atypical bacteria were reported as not detected or detected, and resistance genes were reported as positive. In the case of positive results, semiquantitative values expressed in DNA copies/mL were also reported for each pathogen detected. All steps from nucleic acid extraction to the final detection of pathogens were carried out in an automated manner. The sample swab included in the kit was used to dispense the appropriate amount of mini-BAL or ETA into the cartridge according to the manufacturer’s instructions. Briefly, approximately 200 μL of the sample was collected using a flocked swab and transferred to a sample injection vial. It was then mixed with the provided sample buffer. This solution was then loaded into the FilmArray pouch, which in turn was loaded into the FilmArray platform. The preparation of each cartridge required approximately 2 min of hands-on time, while the run time was approximately 1 h and 15 min , 15.Coinfection was defined as the identification of a respiratory tract pathogen using the FA-PNEU or microbiological cultures of respiratory samples from patients with severe COVID-19 infection.th and 75th percentiles. Overall concordance was calculated as  × 100, positive concordance was calculated as  × 100), and Cohen's kappa coefficient with 95% confidence intervals was estimated with FA-PNEU and conventional culture results. Sensitivity, specificity, positive and negative predictive values (PPVs and NPVs), along with 95% confidence intervals, were calculated by comparing the results between conventional culture and the FA-PNEU only for bacterial pathogens present in the molecular panel. Performance was measured considering bacterial culture as the gold standard reference method. We did not perform concordance analysis or qualitative agreements for atypical bacteria or viruses. The association between the presence of coinfection and the inflammatory response was measured by the Kruskal–Wallis test. Data were entered into a Microsoft Excel database and analyzed using SPSS software version 26.0.Categorical variables were expressed as frequencies and percentages. Continuous variables are presented as medians and 25During the study period, 1289 patients admitted to the ICU with severe COVID-19 were screened, of which 149 met the inclusion criteria  from 149 patients, of which 110 samples were processed through the FA-PNEU and 149 through conventional culture. Of the 110 samples tested by both methods, a total of 27 samples (24.54%) were detected by the FA-PNEU vs. 19 samples (17.27%) detected by culture. In total, the FA-PNEU identified 40 microorganisms and cultured 23. Table Aspergillus flavus, which was assumed to be caused by contamination by medical staff), and 9 samples were FA-PNEU-positive and culture-negative . The two bacteria most frequently detected by the FA-PNEU were S. aureus (37.5%) and S. agalactiae (20%), and those detected by culture were S. aureus (34.78%) and Klebsiella pneumoniae (26.08%). Atypical bacteria were not detected by the FA-PNEU. Two samples with rhinovirus/enterovirus were detected by the FA-PNEU.Of the 110 samples that underwent both techniques, 18 samples were positive for both methods, 82 samples were negative for both techniques, 1 sample was culture-positive with a negative FA-PNEU result  were polymicrobial compared with 4/19 (21.05%) culture-positive samples. The most common combinations found were S. aureus were methicillin-resistant (MRSA). In six samples, the FA-PNEU detected the MecA/C/MREJ resistance mechanism in S. aureus, which was not detected by conventional cultures. None of the K. pneumoniae isolates had CTX-M, KPC, NDM, Oxa48-like, VIM or IMP by the FA-PNEU or extended-spectrum beta-lactamases (ESBL) or carbapenemases by culture. In conventional culturing, one patient was positive for Streptococcus pneumoniae resistant to ceftriaxone, which was not detected by the FA-PNEU.According to the FA-PNEU, 40% of the detected Enterobacter cloacae and S. aureus. Negative predictive values (NPVs) were high (99.1%–100%). Regarding resistance mechanisms, MecA/C/MREJ had a specificity of 94.55% and an NPV of 100%.Table 5 CFU, 84.21% had a count of ≥ 105 copies/mL in the FA-PNEU result; of the negative cultures, 40.9% had microorganisms with a count < 105 copies/mL in the FA-PNEU result. The 10 samples taken by mini-BAL were negative in both cultures and the FA-PNEU.Regarding the quantitative agreement, of the microorganisms in the cultures of ETA samples with > 10p = 0.219) and culture-positive individuals . The inflammatory response tests showed no significant differences between patients whose samples were positive and negative for both techniques  received at least one dose of empirical antibiotics after LRT samples were taken. The antibiotic regimen most commonly used was ceftriaxone (45.9%), followed by cefepime (31.1%) and ampicillin/sulbactam (23%). Later, when the treating physicians knew the result of the FA-PNEU, in 58 (95.1%) of the 61 patients, the therapy was modified as follows: In 21 (91.3%) of the 23 cases in which the FA-PNEU result was positive, the therapy was changed, and in 37 (97.4%) of the 38 cases in which the FA-PNEU result was negative, the antibiotics were suspended (Table 5.45% recForty-nine 44.55%) of the 110 patients did not receive empiric antibiotics after LRT samples were taken. Of these, the FA-PNEU result was negative in 45 cases, and no antibiotics were started; in the other 4 cases where the FA-PNEU result was positive, antibiotics were started  and S. agalactiae (20%) by the FA-PNEU and S. aureus (34.78%) and K. pneumoniae (26.08%) by culture. Verroken et al. [S. aureus (38.46–60% methicillin-sensitive), H. influenzae (23.07%), and Moraxella catarrhalis (15.38%) were the main pathogens identified. Kreitmann et al. [S. aureus , H. influenzae (38.5%), and S. pneumoniae (23.1%). Kolenda et al. analyzed 99 patients with respiratory samples taken in the absence of mechanical ventilation or during their first 48 h; conventional cultures detected bacterial coinfection in 15%, with S. aureus , H. influenzae (26.66%), and S. pneumoniae (13.33%) being the most prevalent pathogens.The two most frequently detected bacteria were n et al.  reportedn et al.  documentS. aureus was the most prevalent microorganism; second, in the present study, methicillin resistance was higher ; and last, unlike other studies, K. pneumoniae was the second most prevalent microorganism in the current study by culture, with no ESBL or KPC resistance mechanisms.When comparing the aforementioned studies with our work, three aspects are worth highlighting: First, in all the studies, including ours, E. cloacae and S. aureus; the NPV was high (between 99.1% and 100%). Caméléna et al. [S. aureus, as opposed to our study, did have a good PPV (91%). Kolenda et al. [H. influenzae (< 88.4%), and the specificity for S. aureus was 93.5%.Regarding the qualitative agreements between the FA-PNEU and conventional cultures, in our study, we found that the PPV was between 50 and 100% and lower for a et al.  demonstra et al.  reportedS. aureus had a sensitivity of 100%, a PPV of 53.3%, a specificity of 93.1%, and an NPV of 100%, since 6 of the 9 patients with FA-PNEU-positive and culture-negative microorganisms were positive for S. aureus. Moreover, in 6 FA-PNEU samples, the MecA/C/MREJ resistance mechanism was detected and not identified by conventional cultures. Fontana et al. [S. aureus in both BAL  and sputum , with the majority being mecA positive . Although most of the patients were not in the ICU, their results are consistent with our findings.In our study, tests for a et al.  used the5 CFU, 84.21% had a count of ≥ 105 copies/mL in FA-PNEU testing; of the culture-negative samples, 40.9% had microorganisms with a count < 105 copies/mL in FA-PNEU testing. In the study of Kolenda et al. [6 copies/mL using the FA-PNEU; in contrast, among 26 bacteria detected using the FA-PNEU yet culture-negative, 20 (76.9%) had ≤ 105 copies/mL using the FA-PNEU. We can conclude that most positive samples in the FA-PNEU results, with negative cultures, have low DNA copies/mL. These findings raise the following questions: Is it possible that in these cases, it is not strictly a coinfection and rather a contamination by the endogenous flora? What is the clinical impact of this finding? While culturing remains the gold standard in the diagnosis of bacterial respiratory tract infections, it may be difficult to accurately recover all pathogens in clinical samples, as the organisms are in a complex matrix. In addition, culture results would be more affected by the host immune response and prior antibiotic usage. Antimicrobial therapy can impact bacterial growth, leading to negative cultures but to persistent positive FA-PNEU results, which is not able to distinguish dead from viable bacteria. For this reason, we excluded patients who, at the time of respiratory sample collection, had received any dose of empiric antimicrobial therapy. Distinguishing colonizing organisms from pathogens remains a challenge because levels of bacteria below the culture threshold can provide positive results in the FA-PNEU. However, previous studies have described that the bacterial burden could be overestimated by FilmArray compared to culturing [Concerning the quantitative agreement, in our study, microorganisms in cultures of ETA samples with > 10ulturing , 20.In future studies, we will try to give some answers by comparing the results of the cultures and FA-PNEU with samples from the lung microbiome through the extraction of DNA and RNA for sequencing of the same samples for metagenomics and metatranscriptomics, which will allow us to determine the functional profiles of the virulence and resistance genes of microorganisms and differentiate expressed human genes. These strategies that examine the host response provide opportunities to rethink what defines true pneumonia and lung coinfection, so some propose a reconceptualized view of pneumonia, in which the development of pneumonia is believed to result from disruption of the complex homeostasis of a microbial ecosystem interacting with multiple complex growth conditions .5 copies/mL, the inflammatory response was lower than that in patients with positive FA-PNEU results with a count of > 105 copies/mL, which could suggest that these patients have more colonization than infection. There are few studies evaluating whether acute phase reactants are useful as predictors of coinfection in patients with COVID-19. Bolker et al. [We did not observe differences between inflammatory biomarker levels and pulmonary coinfection defined by the samples that were positive and negative for both techniques, the FA-PNEU and culturing. However, although the sample was not enough to make statistical comparisons, we found that in culture-negative patients with positive FA-PNEU results with a count of < 10r et al.  found thr et al. , in a reWe did not find that coinfection by culture or by the FA-PNEU was associated with an increase in mortality. Another Latin American study conducted by Soto et al. evaluated ninety-three hospitalized patients with a diagnosis of COVID-19 who were analyzed with the FA-PNEU. Coinfection was evidenced in 38 (40.86%) cases, and no association with mortality was found  .Based on only a few studies without defined information on sampling strategies, a bacterial or fungal coinfection rate of 8% in COVID-19 patients was estimated, but 72% of all these reported COVID-19 patients received (empiric broad-spectrum) antibiotic therapy . In a st4 CFU/mL for a mini-BAL fluid sample or ≥ 105 CFU/mL for an ETA sample were considered; we did not take into account growth of < 105 for an ETA sample to avoid overestimating the prevalence of coinfection. Fifth, the BioFire® FilmArray® Pneumonia Panel technique has been validated for ETA samples [A limitation of our study is that the samples were collected either by endotracheal aspirate or by mini-BAL and not through BAL. However, we did this for several reasons: First, the latest US guidelines recommend noninvasive sampling  with semiquantitative cultures to diagnose VAP and non-VAP; there is no evidence that invasive microbiological sampling with quantitative cultures improves clinical outcomes compared with noninvasive sampling with either quantitative or semiquantitative cultures. Second, noninvasive sampling can be performed more rapidly than invasive sampling, with fewer complications and resources; it is our usual practice due to the lack of availability of pulmonary professionals 24 h a day, and this applies even more during the COVID-19 pandemic. Third, the ETA samples were collected properly, and the respiratory samples were not processed when a quality level of 0 or 1 was detected by the Murray/Washington criteria, based on the number of squamous cells and neutrophils per field. Fourth, only results with growth ≥ 10 samples There are several strengths in this study. First, the high NPV of the FA-PNEU was demonstrated; therefore, we can conclude that if we find a negative result, bacterial coinfection is practically excluded. Novy et al. proposed an algorithm for the rational use of the FA-PNEU in critically ill ventilated COVID-19 patients; this would allow 65.6% of antibiotic sparing for bacterial coinfection and better adequacy of empirical antibiotic therapy . Second,S. aureus. Concerning qualitative agreements between the FA-PNEU and conventional cultures, we found that the PPV ranged between 50 and 100% and was lower for E. cloacae and S. aureus, with a high NPV (between 99.1 and 100%). The overall concordance was 90.1%, and it was between 92.7 and 100% when stratified by microorganisms.Bacterial coinfection in critically ill COVID-19 patients was present in 24.54% of samples tested by the FA-PNEU and in 17.27% of samples tested by conventional culture. The most frequently isolated microorganism was


The purpose of this study was to determine the psychometric validity of the Visual Function Index (VF-14) for use by patients with Leber hereditary optic neuropathy (LHON).Rasch analysis was conducted in two stages using data for 196 individuals  carrying one of the three primary LHON mutations and affected by vision loss. In stage 1, scale unidimensionality, scale-to-sample targeting, response category threshold ordering, item fit statistics, local dependency, and reliability were assessed. In stage 2, iterative post-hoc revisions of the VF-14 structure (VF-14R) were applied and psychometrically re-evaluated.Issues identified with the VF-14 included disordered response thresholds (12/14 items), local dependency (10/91 pairwise dependencies), and evidence of multidimensionality. However, the distribution of person estimates and item thresholds were fairly well matched, only one item showed misfit to the Rasch model, and there was good reliability (Person Separation Index 0.84). Rasch-informed VF-14 revisions included removing both driving items and the misfitting sports item, rescoring response options across all items by merging two response categories, and accounting for the dependency between two reading items. The VF-14R demonstrated improved psychometric validity.Clinicians and researchers using the VF-14 with LHON patients should be aware of its limitations. Compared to the original version, the proposed Rasch-based structure of the VF-14R appears to offer improved psychometric performance and interpretation of vision-related activity limitation.The original version of the VF-14 exhibits several limitations that undermines its psychometric validity as a patient-reported outcome measure for patients with LHON. Participants were included if they were confirmed to have one of the three primary LHON mutations and were affected by vision loss because of LHON. Asymptomatic carriers and individuals with vision loss from other causes or LHON-plus syndrome were excluded. Details regarding recruitment have been described in a previous study.The VF-14 was administered to study participants by three investigators via telephone. Participants were asked to give answers that incorporated the use of reading glasses, but not visual aids such as magnifiers.The VF-14 asks respondents to rate their ability in performing 14 vision-dependent activities in everyday life and the degree of difficulty for each activity.http://www.R-project.org). The psychometric validity of the VF-14 was assessed by Rasch analysis using RUMM2030 Plus .Statistical analysis of demographic data was performed with R: A language and environment for statistical computing , thereby permitting the use of parametric statistical techniques.Rasch analysis of the VF-14 was conducted in two stages. In stage 1, response category threshold ordering, item fit statistics, local dependency, scale-to-sample targeting, scale reliability, and scale unidimensionality were assessed. In stage 2, iterative post-hoc revisions of the VF-14 structure (VF-14R) were applied and psychometrically re-evaluated.One hundred ninety-six individuals  with LHON completed the VF-14. The median age of respondents was 41 years , with median disease duration of nine years . Most respondents (67.3%) harbored the m.11778G>A LHON mutation, followed by m.3460G>A mutation (17.9%) and the m.14484T>C mutation (14.8%).The median VF-14 score was 17.1 , using the scoring system out of 100 recommended by the developers. Only 10 respondents provided a complete response for all 14 items without a “not applicable” response. Four items had complete responses . Of the 10 items that had incomplete responses, both driving items (“driving during the day” and “driving at night”) had the lowest response rate (5.1% for both items), indicating that most respondents did not drive, either because they had stopped driving for reasons other than their vision or had never learned to drive for whatever reason. Other items with incomplete responses included “participation in sports” (33.5% incomplete responses), “playing games” (33.0%), “doing fine handwork” (24.9%), and “cooking” (9.1%). The distribution of responses across all 14 items are summarized in 2(28) = 96.67, P < 0.01). Only two items (“seeing steps” and “doing fine handwork”) showed ordered response category thresholds. For the items that showed disordered response category thresholds, there was frequent nonfunctioning of the middle response categories, especially the “a little difficulty” category , indicating that that the VF-14 appeared to be measuring more than one underlying construct.Post-hoc revisions to the structure of the VF-14 (VF-14R) were made in response to the anomalies identified in stage 1. Both driving items were removed in response to the very low response rates. The “participation in sports” item was also removed due to significant misfit to the Rasch model. In order to correct the disordered response thresholds, a post-hoc rescore was applied across all items where the response categories “moderate difficulty” and “a little difficulty” were merged. Additionally, the “reading small print” and “reading newspaper/book” items were merged into one “super item” to account for the local dependency exhibited between them, because both items were conceptually similar.2(20) = 31.23, P = 0.052). All items displayed ordered response categories, and none of the items were misfitting. Only 1/45 item pair dependencies were identified. However, the item pair (“cooking” and “playing games”) dependency was borderline , and the conceptual link between these items is less clear. The Person-Item Threshold Distribution of the VF-14R continued to show reasonable spread of estimated person ability and item difficulty along the logit continuum, with a minor floor effect. There was good reliability with a PSI of 0.852. Unidimensionality of the VF-14R was confirmed (2.8% of paired t-tests significant at the P < 0.05 level).After these post-hoc adjustments, the VF-14R was psychometrically re-evaluated, and satisfactory fit to the Rasch model was demonstrated . Differences in the demographic background of individuals with LHON compared to individuals with cataracts, likely explain the incomplete responses. The peak age of onset of vision loss in LHON is between 15 and 35 years, with a subset of patients under the age of 12 years developing a form of childhood-onset LHON.,For PROMS to be effective as an outcome measure in clinical trials, they have to capture the disease characteristics that matter to the patient and must be reliable and valid.A weakness of the VF-14 in its original format is the conversion of raw scores for individual items into an average score out of 100. This assumes that each item contributes equally to the final score, and that the interval between each response category is uniform across all the categories and for all items. A key strength of this study was the use of Rasch analysis to overcome this limitation by calculating item difficulty in relation to person ability and adjusting the overall scores accordingly, allowing comparison of measures and interpretation of changes in scores when the logit scores are used. Previous studies have found that Rasch-informed revisions to the VF-14 are more sensitive to change than the original VF-14, when used in longitudinal studies of patients with cataracts who undergo surgery.,Limitations of this study relate to the study participants. Individuals with LHON were recruited from large university clinics in three European countries. Patients seen in these clinics may have more complex disease or severe disease because of referral bias. Except for one respondent of Asian descent, all respondents were white. The experiences of these respondents residing mainly in Europe may differ from those elsewhere in the world .In summary, the VF-14 in its original format exhibits several limitations that undermines its psychometric validity as a PROM for assessing vision-related activity limitation in individuals with LHON. These limitations likely stem from differences in the clinical and demographic characteristics of individuals with LHON compared to those with cataracts, leading to problems with disordered response category thresholds, item misfit, and scale multidimensionality. Rasch informed post-hoc revisions of the VF-14 can improve the psychometric validity of the PROM. However, these revisions do not overcome issues relating to PROM content. Future studies should evaluate the sensitivity of other established scales for conditions that have similar clinical and demographic characteristics to individuals with LHON, such as people with low vision, or focus on the development of a LHON-specific PROM that captures the disease characteristics that matter most to LHON patients.